Microinjection of cryoprotectants for preservation of cells

ABSTRACT

A preservation method for biological material having cell membranes includes microinjecting the cell membranes with sugar; preparing the cells for storage; storing the biological material; and recovering the stored biological material from storage. Carbohydrate sugars such as trehalose, sucrose, fructose, dextran, and raffinose, may be used as bio-protective agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. ProvisionalApplication No. 60/204,877, filed May 16, 2000.

FIELD OF THE INVENTION

[0002] The present invention relates to the preservation of biologicaltissue using microinjection of intracellular protective agentscontaining sugar to preserve cells by freezing and/or drying.

BACKGROUND OF THE INVENTION

[0003] In recent years, chemotherapy and radiation therapy of patientswith cancer has been increasingly successful and sustained remissionshave been achieved. However, the chronic side effects of these therapiesto the reproductive systems of long-term survivors is of particularconcern. These effects for women include depletion of ovarian germ cellsand sterility. Due to the potential loss of future fertility of thoseexposed to cancer therapy, a need for oocyte banking has developed.Oocyte freezing, when combined with in vitro fertilization, may bebeneficial to women desiring future fertility who are anticipating lossof gonadal function from extirpative therapy, radiation, orchemotherapy. Oocyte freezing may also provide a possible alternative tohuman embryo freezing, thus avoiding many of the legal and ethicalproblems encountered in embryo freezing.

[0004] The first successful cryopreservation of human embryos wasachieved in 1983 and embryo freezing is now a routine procedure. Incontrast, very limited success has been reported with cryopreservationof human oocytes. Only five successful pregnancies have been reportedwith more than 1500 cryopreserved oocytes. Therefore, the currentmethods of freezing are still considered experimental and novelapproaches are needed to overcome the difficulty encountered bycryopreservation of the human oocyte.

[0005] Traditional cryopreservation techniques include penetratingcryoprotectants at concentrations of 1 to 2M with, for example, dimethylsulfoxide (DMSO), glycerol, or ethylene glycol, followed by a slowfreezing rate (0.3 to 0.5° C./min). Typically, oocytes are damaged dueto long-term exposure to deleterious freezing conditions, includingexcessive dehydration and high electrolyte concentrations. Analternative approach, called vitrification (i.e. formation of glassymaterial without crystallization of ice, uses high concentrations ofcryoprotectant mixtures (6 to 8M) followed by rapid cooling in order toavoid the lethal effects of freezing on oocytes.

[0006] Though an attractive alternative, vitrification procedures sufferfrom the toxic and osmotic effects of high cryoprotectant concentrationon sensitive cells. Neither of these two approaches (slow freeze-thawand rapid vitrification) has resulted in a reliably successful outcomefor cryopreservation of human oocytes. Thus, there is a need for areliable technique for human oocyte storage. In order to provide thepreservation of mammalian cells necessary for application of livingcells as a therapeutic tool in clinical medical care, new protocols forpreserving living nucleated cells using low levels of non-toxicpreservation agents and having simple procedures applicable to a varietyof cells must be developed.

SUMMARY OF THE INVENTION

[0007] The purpose of the present invention is to allow the storage ofliving cells in a dormant state and the subsequent recovery of the cellsto an active state. This method involves microinjecting into thecytoplasm of a cell a protective agent that is substantiallynon-permeating with respect to mammalian cell membranes and thatmaintains the viability of the cell such that it can be stored in atemporarily dormant state and substantially restored to an active state.The microinjected cell is subjected to conditions that cause it to entera dormant state and is stored in this dormant state. The stored cell canbe subsequently restored to an active state. This method has theadvantage of allowing any mammalian cell to be stored until it is neededunder conditions that cause minimal, if any, adverse side-effects in thecell.

[0008] Thus, the invention, in some embodiments, provides a method forpreserving living cells that begins with microinjecting a protectiveagent containing an effective sugar into the cell, preferably an oocyte.Other preferred cells that may be preserved include differentiatedcells, such as epithelial cells, neural cells, epidermal cells,keratinocytes, hematopoietic cells, melanocytes, chondrocytes, B-cells,T-cells, erythrocytes, macrophages, monocytes, fibroblasts, or musclecells; and undifferentiated cells, such as embryonic, mesenchymal, oradult stem cells. In one preferred embodiment, the differentiated cellsremain differentiated after they are recovered from a frozen or driedstate, and the undifferentiated cells remain undifferentiated after theyare recovered. The cells can be haploid (DNA content of n; where “n” isthe number of chromosomes found in the normal haploid chromosomes set ofa mammal of a particular genus or species), diploid (2n), or tetraploid(4n). Other cells include those from the bladder, brain, esophagus,fallopian tube, heart, intestines, gallbladder, kidney, liver, lung,ovaries, pancreas, prostate, spinal cord, spleen, stomach, testes,thymus, thyroid, trachea, ureter, urethra, or uterus. The cells may befrom a human or non-human mammal, such as a monkey, ape, cow, sheep,big-horn sheep, goat, buffalo, antelope, oxen, horse, donkey, mule,deer, elk, caribou, water buffalo, camel, llama, alpaca, rabbit, pig,mouse, rat, guinea pig, hamster, dog, or cat.

[0009] The method of the invention may advantageously use low levels,less than or equal to about 6, 5, 4, 3, 2, or 1 M, or even less thanabout 0.4 M of preservation agent, and may use a sugar alone as thepreservation agent, or sugar in combination with a conventionalcryoprotectant, or in combination with other intracellular sugars orextracellular sugars. More preferably, the cytoplasmic concentration ofthe sugars is less than 0.3, 0.2, 0.1, 0.05, or 0.01 M aftermicroinjection and before freezing or drying of the cell. Theextracellular concentration of the sugars is preferably less than 0.3,0.2, 0.1, 0.05, or 0.01 M after dilution into a liquid medium containingthe cell. If the cell is grown on a solid support, such as an agarplate, the concentration of the sugars in the preservation agent that iscontacted with the cell is preferably less than 0.3, 0.2, 0.1, 0.05, or0.01 M. In other preferred embodiments, the final concentration ofextracellular sugar in the medium containing the cell is at least 2, 3,4, 5, or 10-fold greater than the cytoplasmic concentration ofintracellular sugar after microinjection and before freezing or dryingof the cell. The intracellular and extracellular preservation agents maybe the same or different molecular species.

[0010] Preferred protective agents include sugars such asmonosaccharides, disaccharides, and other oligosaccharides. Preferably,the agent is substantially non-permeable such that at least 50, 60, 70,80, 90, or 95% of the agent does not migrate across the plasma membraneinto or out of the cell, by active or passive diffusion. Preferredsugars have a glass transition temperature of the maximallyfreeze-concentrated solution (Tg′) that is at least −60, −50, −40, −30,−20, −10, or 0° C. Examples of such sugars are those listed in FIG. 8.The Tg′ of other sugars may be routinely determined using standardmethods such as those described by Levine and Slade (J. Chem. Soc.,Faraday Trans. 1, 84:2619-2633, 1988). The sugar or conventionalcryoprotectant with a Tg′ below −50° C. can be combined with a sugarwith a Tg′ above −50° C. such that the resulting mixture has a Tg′ of atleast −60, −50, −40, −30, −20, −10, or 0° C., and this mixture is usedfor cryopreservation.

[0011] Suitable monosaccarides include those that have an aldehyde group(i.e., aldoses) or a keto group (i.e., ketoses). Monosaccharides may belinear or cyclic, and they may exist in a variety of conformations.Other sugars include those that have been modified (e.g., wherein one ormore of the hydroxyl groups are replaced with halogen, alkoxy moieties,aliphatic groups, or are functionalized as ethers, esters, amines, orcarboxylic acids). Examples of modified sugars include α- orβ-glycosides such as methyl α-D-glucopyranoside or methylβ-D-glucopyranoside; N-glycosylamines; N-glycosides; D-gluconic acid;D-glucosamine; D-galactosamine; and N-acteyl-D-glucosamine. In otherpreferred embodiments, the preservation agent is an oligosaccharide thatincludes at least 10, 25, 50, 75, 100, 250, 500, 1000, or more monomers.The oligosaccharide may consist of identical monomers or a combinationof different monomers. Other suitable oligosaccharides include hydroxylethyl starch, dextran, cellulose, cellobiose, and glucose. Othersuitable preservation agents include compounds that contain a sugarmoiety and that may be hydrolytically cleaved to produce a sugar. Stillother suitable preservation agents include glycoproteins andglycolipids, which preferably have been modified by the addition of 1,2, 3, 4, 5 or more sugar moieties derived from sugars with a Tg′ of atleast −60 −−40, −30, −20, −10, or 0° C. or with a molecular weight of atleast 120 daltons. By “sugar moiety” is meant a protective sugar thatincludes a group that can be bonded to another compound. For example, areactive group—such as an alcohol, primary amine, or secondary amine—ina sugar can react with a compound, forming a product that includes thesugar moiety.

[0012] Another suitable extracellular preservation agent is a lectin orany protein that can non-covalently or covalently bind to a sugar thatforms part of a cell-surface glycoprotein or glycolipid. This bindingmay stabilize the cellular membrane during storage of the cell.

[0013] Examples of other cyroprotectants that may be used in the methodsof the present invention include sugars, polyols, glycosides, polymers,and soluble proteins with a molecular weight of at least 120 daltons. Asillustrated in FIGS. 8 and 9, compounds with higher molecular weightstend to promote glass formation at a higher temperature than thatpromoted by smaller compounds, allowing the cells to be stored at ahigher storage temperature.

[0014] After treatment with the microinjected protective agent and,optionally, the external protective agent, the cell is then prepared forstorage. In general, the cell may be prepared for storage by freezingand/or drying. Plunge freezing, vacuum drying, air drying, as well asfreeze drying techniques may be employed. Typically, oocytes are cooledat a rate of 0.1 to 10° C./min, preferably, between 0.3 and 5° C./min,and, more preferably, between 0.5 and 2° C./min, inclusive. Somaticcells are cooled at a rate between 0.1 and 200° C./min, preferably,between 0.5 and 100° C./min, and, more preferably, between 1 and 10°C./min or 10 and 50° C./min, inclusive. The cells are cooled to a finaltemperature of at least −60, −50, −40, −30, −20, −10, 0, 10, or 20 ° C.(in order of increasing preference). In another preferred embodiment,the preservation agent inhibits or prevents the nucleation or growth ofintracellular ice during freezing of the cells.

[0015] Extracellular preservation agents may reduce the osmotic shock tothe cells that potentially results from the addition of an intracellularpreservation agent. Additionally, extracellular preservation agents maystabilize plasma membranes and provide mechanical strength to the cellsduring freezing or drying.

[0016] Once the cell is prepared for storage, it is stored in a mannerappropriate to its preparation. Frozen cells can be stored at cryogenictemperatures and dried cells can be dry stored at ambient or othertemperatures as appropriate. Recovery of stored cells is geared to themethod of their preparation for storage. Dried cells are rehydrated, andfrozen cells are thawed. Preferably, at least 25, 35, 50, 60, 70, 80,90, 95, or 100% of the recovered cells are viable. Cell viability may bemeasured using any standard assay, such as a “live/dead” assay using thegreen dye calcein-AM to indicate viable cells and the red dye ethidiumhomodimer to indicate dead cells, according to the manufacturer'sprotocol (Molecular Probes, Inc.). In another preferred embodiment, atleast 5, 10, 15, 25, 35, 50, 60, 70, 80, 90, or 95% of the recoveredoocytes may be fertilized using standard in vitro fertilizationtechniques (see, for example, Summers et al., Biol. Reprod. 53:431-437,1995). Preferably, the fertilized oocytes develop into 2-cell stageembryos, 4-cell stage embryos, morula-stage embryos, blastocyst-stageembryos, fetal-stage embryos, or viable offspring.

[0017] By “embryo” is meant a developing cell mass that has notimplanted into the uterine membrane of a maternal host. Hence, the term“embryo” may refer to a fertilized oocyte, a pre blastocyst stagedeveloping cell mass, or any other developing cell mass that is at astage of development prior to implantation into the uterine membrane ofa maternal host and prior to formation of a genital ridge. An embryo mayrepresent multiple stages of cell development. For example, a one cellembryo can be referred to as a zygote; a solid spherical mass of cellsresulting from a cleaved embryo can be referred to as a morula, and anembryo having a blastocoel can be referred to as a blastocyst.

[0018] By “fetus” or “fetal” is meant a developing cell mass that hasimplanted into the uterine membrane of a maternal host. A fetus may havedefining features such as a genital ridge which is easily identified bya person of ordinary skill in the art.

[0019] In another aspect, the invention provides a method of culturingmammalian cells in vitro by incubating the cells in a hypertonic mediahaving an osmolarity of greater than 300, 310, 320, 330, 340, 350, 360,370, 380, or more mosm. Preferably, the media includes one or more ofthe components listed in FIG. 7 or one or more cryopreservation agentsof the present invention. Preferably, the media contains nutrients suchas amino acids, sugars, lactate, or pyruvate. This media may be used toculture any mammalian cell, including the preferred cells listed above.In various preferred embodiments, this media is used to culture cellsbefore, during, or after cryopreservation.

[0020] The present invention provides a number of advantages related tothe cryopreservation of cells. For example, these methods may begenerally applied to the preservation of any cell from any mammal. Thesecells may be stored in a frozen or dried state for any length of timeuntil they are needed. Additionally, these cryopreservation methodsinvolve the use of relatively low concentrations of non-toxicpreservation agents that cause minimal, if any, adverse side-effects inthe stored cells. Moreover, the preservation agents reduce or eliminatethe formation of intracellular ice during freezing which would otherwisedamage the cells. If desired, both intracellular and extracellularpreservation agents may be used to reduce the osmotic pressure caused bythe addition of an intracellular preservation agent to the cells.Extracellular preservation agents may also stabilize plasma membranesand provide mechanical strength to the cells during freezing or drying.Furthermore, the present invention may enable a higher storagetemperature (preferably, greater than −60° C.) compared to conventionalcyroprotectants (typically less than −80° C.) due to the high Tg′ ofsugars.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The invention will be more fully understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which:

[0022]FIG. 1 is a flow chart showing steps in the method of theinvention.

[0023]FIG. 2 is a schematic flow diagram listing one embodiment of thecyropreservation protocol of the present invention. This protocol may bemodified by one skilled in the art for the preservation of other cellsusing other cyropreservation agents, cooling rates, dilution steps, andmedias.

[0024]FIG. 3A is a graph showing the volume of mouse oocytes in isotonicDMEM/F-12 media prior to microinjection, isotonic media with 0.1 Mextracellular trehalose prior to microinjection, and isotonic media with0.1 M extracellular trehalose after microinjection of 0.1 M trehalose.FIGS. 3B-3D are phase-contrast microscopy pictures of the oocytes undereach of the three conditions listed above.

[0025] FIGS. 4A-4D are phase-contrast microscopy pictures of humanoocytes in isotonic DMEM/F-12 media prior to microinjection, isotonicmedia with 0.15 M extracellular trehalose prior to microinjection,isotonic media with 0.15 M extracellular trehalose duringmicroinjection, and isotonic media with 0.15 M extracellular trehaloseafter microinjection of 0.15 M trehalose, respectively.

[0026]FIG. 5 is a bar graph illustrating the calibration of a syringeused for microinjection of a cyropreservation agent of the invention.

[0027]FIG. 6 is a phase diagram of DMSO and trehalose.

[0028]FIG. 7 is a table listing the components of HTF; modified HTF,isotonic; and modified HTF, hypertonic medias.

[0029]FIG. 8 is a table listing the percentage of metaphase II mouseoocytes with or without intracellular trehalose that were fertilized anddeveloped into blastocysts while being cultured in modified HTF,isotonic or modified, HTF, hypertonic media.

[0030]FIGS. 9A and 9B are a set of graphs showing the percent survivalfor metaphase II mouse oocytes with 0 or 0.10-0.15 M intracellulartrehalose in the presence of various concentrations of extracellulartrehalose.

[0031]FIG. 10 shows the survival of cooled metaphase II mouse oocytesafter overnight culture

[0032]FIG. 11 shows the survival of cooled human oocytes after overnightculture.

[0033]FIG. 12A is a graph of a set of curves showing the normalizedwater volume in oocytes as a function of temperature for differentcooling rates. FIG. 12B is a graph showing the calculated dehydrationtime as a function of temperature. The dehydration time is defined asthe time necessary for an oocyte to shrink in volume by 50% at a giventemperature.

[0034]FIG. 13 is graph of a set of curves showing the cumulativeincidence of intracellular ice as a function of temperature in thepresence and absence of preservation agents.

[0035]FIG. 14 is a table listing the molecular weight and glasstransition temperature for sugars with a glass transition temperaturegreater than −55° C. (Levine and Slade, J. Chem. Soc., Faraday Trans. 1,84:2619-2633, 1988). Many of these sugars are commercially availablefrom sources such as Sigma and British Sugar.

[0036]FIG. 15 is a graph showing the monotonic relationship between Tg′and molecular weight of several sugars and traditional cryoprotectants.

DETAILED DESCRIPTION

[0037] A method for preserving biological tissue of the invention,illustrated in FIG. 1 and FIG. 2, starts with the selection or isolationof the cells or tissue to be preserved 10. While the method of theinvention may be used for preservation of any biological material havinglipid membranes, it is most useful for preservation of living nucleatedcells and, in particular, otherwise difficult to preserve mammaliancells such as oocytes.

[0038] Oocytes can be obtained by isolating ovaries and releasing theoocytes. The oocytes are transferred to hyaluronidase, an enzyme thatbreaks down extra cells. The oocytes are then washed twice in a twoseparate mixtures of PBS (phosphate buffered saline) and BSA (bovineserum albumin). Oocytes are then transferred to HEPES-bufferedDulbecco's Modified Eagle Medium/Nutrient F-12 (DMEM/F-12) mixture(Gibco) covered with embryo-tested mineral oil (Sigma), or any othersuitable media. The DMEM/F12 media is preferably supplemented with 4mg/mL BSA. If desired, the oocytes may also be incubated withextracellular sugar at the same concentration as the amount planned formicroinjection. For example, to inject 0.1 M sugar, oocytes may beequilibrated in DMEM/F-12 with 0.1 M sugar. As illustrated in FIGS.3A-3C, the hyperosmoticity of the external DMEM/F-12+sugar solutioncauses mouse oocytes to shrink. This decrease in cell volume may bequantitated by visually measuring the diameter of the cells usingphase-contrast microscopy. The decrease in cell volume facilitates thedetermination of how much sugar is subsequently microinjected into theoocytes. For example, the swelling of cells during microinjection totheir initial isotonic volume (i.e., the cell volume prior toequilibration with external sugar) indicates that the concentration ofinjected sugar is close to that of the extracelluar sugar concentration(FIGS. 3A-3D). Similar results were obtained when human oocytes wereincubated in 0.15 M extracellular trehalose causing their volume todecrease and then injected with trehalose until the volume of theoocytes returned to their initial volume in isotonic media, indicatedthat 0.15 M trehalose had been injected (FIGS. 4A-D). Alternatively, theoocytes may be optionally equilibrated with any other substantiallynon-permeable solute, such a NaCl, to decrease their cell volume priorto microinjection. This initial decrease in cell volume may result in asmaller final volume of the microinjected oocytes compared to oocytesnot incubated in a hypertonic media prior to microinjection. Thissmaller final volume may minimize any potential adverse effect from theswelling of the oocytes. This general procedure for the preparation ofcells for microinjection may also be used for other cell types.

[0039] The target cells are then microinjected 30 with abio-preservation agent. Microinjection equipment and procedures are wellcharacterized in the art and microinjection equipment known for use ininjecting small molecules into cells may be used with the invention. Inan exemplary microinjection step, oocytes can be microinjected at apressure of 10 psi for 30 milliseconds. Another example of a standardmicroinjection technique is the method described by Nakayama andYanagimachi (Nature Biotech. 16:639-642, 1998).

[0040] If desired, the syringe used for microinjection of thecyropreservation agent may be calibrated prior to injection (FIG. 5).For this calibration, a microdroplet of dimethylpolysilaxane wasinjected with DMEM/F-12. Preferably, the microdroplet has a similar sizeas that of the cells that will be microinjected. More example,microdroplets with a diameter of 80-100 were used to calibrate a syringefor the microinjection of oocytes. To measure different injectionvolumes from a given injection pipette, several microdroplets wereinjected. Images of each microdroplet were taken using phase-contrastmicroscopy before and after injection to calculate the increase in thevolume of the droplets. Because the microdroplets are almost perfectspheres, the droplet volumes were reliably calculated using the diameterof the droplets. Additionally, the syringe was calibrated by injecting afluorescent sugar, oregon-green-(og) labeled dextran (Molecular Probes,Inc.), into microdroplets and measuring the total fluorescent intensityof the droplets using a ccd camera. The amount of injected og-labeleddextran was determined based on the fluorescent intensity using acalibration curve of the fluorescent intensity of various amounts ofog-labeled dextran. Additionally, a mixture of 80 ,μL DMEM/F-12 and 20,μL og-labeled dextran was injected into oocytes using themicroinjection/micromanipulator. The fluorescence intensity inside theoocytes was measured using a ccd camera, and the amount of injectedog-labeled dextran was determined using a standard curve, as describedabove.

[0041] To achieve a desired cytoplasmic concentration of sugar aftermicroinjection and before any concentration of the intracellular sugardue to freezing or drying of the cell, either the volume of preservationagent that is injected into the cell, the initial concentration of thesugar in the preservation agent, or both can be adjusted. For example,to achieve a relatively high cytoplasmic concentration of sugar aftermicroinjection, a relatively large volume of preservation agent may beinjected or a relatively high concentration of sugar may be injected.Alternatively, if a lower cytoplasmic concentration of sugar is desired,the volume of preservation agent that is injected may be decreased, theconcentration of the sugar in the preservation agent may be reduced, orboth changes may be made. The volume of preservation agent that isinjected may be chosen so that the volume is not so large that theresulting increase in the volume of the cytoplasm causes the cell tolyse. Additionally, the volume of preservation agent may be chosen sothat it is not too small to be accurately measured and injected.

[0042] Similarly, to achieve a desired extracellular concentration ofsugar after dilution into a liquid medium containing the cell, eitherthe volume of preservation agent that is added to the medium, theinitial concentration of the sugar in the preservation agent that isadded to the medium, or both can be adjusted. Thus, the extracellularconcentration of sugar may be increased by adding a larger volume or amore concentrated solution to the liquid medium. For a preservationagent that is added to a cell grown on a solid medium, such as agar, thedesired extracellular sugar concentration can be achieved by contactingthe cell with a preservation agent containing sugar at the desiredconcentration.

[0043] A bio-preservation agent useful in this process includes anychemical that has cryo-protective properties and is ordinarilynon-permeable. In particular, the bio-preservation agent can includesugars either alone or mixed together with other traditionalbio-preservation agents. Carbohydrate sugars such as trehalose, sucrose,fructose, and raffinose, may be microinjected to concentrations lessthan or equal to about 1.0 M, and more preferably, less than or equal toabout 0.4 M. In another preferred embodiment, the concentration isbetween 0.05 and 0.20 M, inclusive. Additionally, an extracellular sugaror traditional bio-preservation agent may be added prior to storage. Ifthe cells were incubated in a hypertonic solution prior tomicroinjection, the substantially non-permeable solute may be allowed toremain in the media after microinjection or may be removed from themedia by washing the cells with media containing a lower concentration,or none, of this solute.

[0044] Certain sugars or polysaccharides which ordinarily do notpermeate cell membranes because they are too large to pass through themembrane have superior physiochemical and biological properties forcryopreservation purposes. While these sugars ordinarily do not permeatecell membranes on their own, using the method of the invention, theseordinarily non-permeating sugars may be microinjected intracellularly toresult in a beneficial effect.

[0045] Non-permeating sugars having a stabilizing or preserving effecton cells that are especially useful as the preservation agent in thepresent method include sucrose, trehalose, fructose, dextran, andraffinose. Among these sugars, trehalose, a non-reducing disaccharide ofglucose, has been shown to be exceptionally effective in stabilizingcell structures at low concentrations. Trehalose is the most preferredsugar for use with the present method. It has an exceptional ability tostabilize and preserve proteins, viruses, and bacteria as well as anunusual ability to form stable glasses at high temperatures. Trehalosehas physicochemical properties for use as an oocyte cell cryoprotectiveagent (CPA) that are far superior to traditional agents. Further,trehalose, contained in many food products, is relatively non-toxic andmay allow for cryopreservation protocols which do not require CPAremoval, resulting in an infusible end product. Sucrose, which hasproperties similar to those of trehalose and which is widely availableand relatively inexpensive, may also be preferred for certainapplications. There are also advantages to using dextran either alone orin combination with other sugars, such as trehalose or sucrose. Dextranhas a very high glass transition temperature (Tg′), but does not havesome of the advantages that are present in sucrose or trehalose, such asthe ability to stabilize biological components. Thus, a dextrantrehalose mixture, or dextran sucrose mixture, may have added benefits.

[0046] The addition of extracellular glycolipids or glycoproteins mayalso stabilize the cell membrane. While not meant to limit the inventionto any particular theory, it is hypothesized that the sugar groups inglycolipids may hydrogen-bond with the hydrophilic head groups ofmembrane phospholipids, stabilizing the membrane againstfreezing-induced stress. Additionally, it is possible that theglycolipids may be incorporated into the lipid bilayer and increase theintegrity of the membrane.

[0047] Following the microinjection of the preservation agent, the cellsare prepared for storage 40. A variety of methods for freezing and/ordrying may be employed to prepare the cells for storage. In particular,three approaches are described herein: vacuum or air drying 50, freezedrying 60, and freeze-thaw 70 protocols. Drying processes have theadvantage that the stabilized biological material may be transported andstored at ambient temperatures.

[0048]FIG. 6 shows a phase diagram of a conventional penetratingcryoprotectant (DMSO) versus a common sugar (trehalose). Typically,oocytes loaded with 1 to 2M DMSO are cooled at a very slow cooling rate(0.3 to 0.5° C./min) to an intermediate temperature (−60° C. to −80° C.)before plunging in liquid nitrogen for storage. As a result of the slowcooling rate, oocytes have ample time to dehydrate and closely followthe equilibrium melting curve (Tm) of the cryoprotectant solution (e.g.,DMSO) down to a temperature range between −300° C. and −50° C. Since thepermeability of the plasma membrane to water decreases exponentially asa function of decreasing temperature at temperatures below −50° C., thecellular dehydration becomes negligible for any practical purposes. Asthe temperature is further decreased to the storage temperature, theunfrozen solution inside the oocytes becomes more concentrated until thetemperature reaches the Tg curve at the point Tg′, and then the solutionbecomes glass. As a result, the fraction of the cellular water whichremains in the cell, crystallizes during further cooling to the storagetemperatures (typically below the glass transition temperature, Tg′).The formation of ice inside the cells is believed to result in celldeath if the fraction of cellular water transformed to ice phasesurpasses a certain limit (usually 5%). On the other hand, the phasediagram of trehalose is such that Tm and Tg curves cross each other at avery high subzero temperature (approximately −30° C.) compared to DMSO(approximately −80° C.). As a result, one can freeze the oocyte, anddehydrate very close to its glass transition temperature while themembrane water permeability is still high, and thereafter plunge inliquid nitrogen to vitrify the sample. The sample can then be stored atthis temperature. This process enables oocytes to overcome the so-called“bottle-neck” effect observed below −50° C. with most conventionalpenetrating cryoprotectants. Beneficial results may also be obtained byapplying extracellular sugars, in addition to the intracellular sugars,to the cell.

[0049] The suspended material can then be stored 90, 100 atcryopreservation temperatures, for example, by leaving the vials in LN₂,for the desired amount of time. Preferably, the cells are stored at atemperature equal to or less than the Tg′ of the cryoprotectant so thatthe cells remain in the glassy state. Preferred storage temperatures areat least 5, 10, 15, 20, 30, or 40° C. below the Tg′. The cells are alsopreferably maintained at a relatively constant temperature duringstorage. Preferably, the storage temperature changes by less than 20,10, 5, or 3° C. during storage. The suspended cells can then berecovered from storage 110 by thawing 120 in a 37° C. water bath withcontinuous, mild agitation for 5 minutes.

[0050] Protocols for vacuum or air drying 50 and for freeze drying 60proteins are well characterized in the art [Franks et al., “MaterialsScience and the Production of Shelf-Stable Biologicals,” BioPharm, Oct.1991, p. 39; Shalaev et al., “Changes in the Physical State of ModelMixtures during Freezing and Drying: Impact on Product Quality,”Cryobiol. 33, 14-26 (1996).] and such protocols may be used to preparecell suspensions for storage with the method of the invention. Inaddition to air drying, other convective drying methods that may be usedto remove water from cell suspensions include the convective flow ofnitrogen or other gases. In one preferred embodiment, the gas used forconvective drying does not contain oxygen which may be deleterious tocertain cells.

[0051] An exemplary evaporative vacuum drying protocol 130 useful withthe method of the invention may include placing 20 μl each into wells on12 well plates and vacuum drying for 2 hours at ambient temperature. Ofcourse, other drying methods could be used, including drying the cellsin vials. Cells prepared in this manner may be stored dry 140, andrehydrated 160 by diluting in DMEM or any other suitable media.

[0052] A method of the invention using freeze drying 60 to prepare thecells for storage 40 begins with freezing 80 the cell suspension. Whileprior art freezing methods may be employed, the simple plunge freezingmethod described herein for the freeze-thaw method may also be used forthe freezing step 80 in the freeze drying protocol.

[0053] After freezing, a two stage drying process 150 may be employed.In the first stage, energy of sublimation is added to vaporize frozenwater. When freeze drying cells, the primary criterion for selecting thetemperature of the primary drying phase is that it must be below theglass phase transition temperature of the freeze concentrated solutionto avoid collapse and undesirable chemical reactions. In general, thehighest possible temperature that will not damage the sample should beused so that sublimation will occur quickly. Typically, the primarydrying occurs at a constant temperature maintained below the glasstransition temperature for the freeze concentrated solution.

[0054] Secondary drying is performed after the pure crystalline ice inthe sample has been sublimated. Secondary drying cannot take placeunless the temperature is raised above the glass phase transitiontemperature of the freeze concentrated solute, however, it is crucialthat the sample temperature does not rise above the collapse temperatureabove which the specimen is believed to mechanically collapse due toviscous flow.

[0055] Freeze dried cells can be stored 140 and hydrated 160 in the samemanner as described above for vacuum drying. Viable cells may then berecovered 170.

[0056] After the recovery of cells from a frozen or dried state, anyexternal cyropreservation may be optionally removed from the culturemedia. For example, the media may be diluted by the addition of thecorresponding media with a lower concentration of cyropreservationagent. For example, the recovered cells may be incubated forapproximately five minutes in media containing a lower concentration ofsugar than that used for cell storage. For this incubation, the mediamay contain the same sugar that was used as the cyropreservation agent;a different cryopreservation agent, such as galactose; or any othersubstantially non-permeable solute. To minimize any osmotic shockinduced by the decrease in the osmolarity of the media, theconcentration of the extracellular cyropreservation agent may be slowlydecreased by performing this dilution step multiple times, each timewith a lower concentration of cyropreservation agent (FIG. 2). Thesedilution steps may be repeated until there is no extracellularcyropreservation agent present or until the concentration ofcyropreservation agent or the osmolarity of the media is reduced to adesired level.

[0057] For cells that divide relatively quickly, such as fibroblasts,the internal concentration of sugar quickly decreases as the sugar isdivided between the mother and daughter cells. Thus, the internalosmolarity of the cells may decrease to a level close to that of atraditional isotonic media, enabling the recovered cells to be culturedin an isotonic media without significant swelling or adverse effectscaused by a difference between the internal and external osmolarity ofthe cells.

[0058] For culturing recovered cells that do not divide or that divideslowly, such as oocytes, using a hypertonic media may prevent or reducethe swelling of the cells that might otherwise occur if they werereturned to an isotonic media. Thus, a hypertonic media for theculturing of certain recovered cells was developed using a two-prongapproach. First, a standard culture medium called HTF (human tubal fluidmedium) was modified to minimize the concentration of electrolytes (seeFIG. 7, components marked with down arrows) and to increase othernutrients and amino acids (see FIG. 7, components marked with uparrows). The osmolarity of this medium was determined using anosmometer. Because the osmolarity of this medium was approximately 285mosm, which is close to the normal internal osmolarity of cells, thismedium is called modified HTF, isotonic. Second, the water content ofthe medium was reduced to equally increase the concentrations of eachcomponent to achieve a final osmolarity of 320 mosm. This medium iscalled modified HTF, hypertonic, (see FIG. 7, last column).

[0059] To test the ability of modified HTF, isotonic and hypertonicmedia to support the fertilization and development of oocytes, thesemedias were used to culture fresh mouse metaphase II oocytes that hadnot been cyropreserved. As illustrated in FIG. 8, control mouse oocyteswithout intracellular trehalose cultured in either modified HTF,isotonic or modified HTF, hypertonic media had a high frequency offertilization (90%) and development to blastocyst-stage (over 85%).Mouse oocytes injected with 0.07M trehalose also showed a high frequencyof fertilization and blastocyst development in modified HTF, hypertonicmedia. At an intracellular trehalose of 0.15 M, the frequencies offertilization and blastocyte formation were reduced, but stillsignificant. If desired, the composition and hypertonicity of theculture medium may be further optimized to increase the number ofblastocysts or viable offspring that are formed. For example, a culturemedium having a higher osmolarity (such as 330, 340, 350, 360, 370, or380 mosm) may better mimic oviductal fluid (which may have an osmolarityof at least 360 mosm) and thus further promote development of viableoffspring (Van Winkle et al., J. Expt Zool. 253:215-219, 1990). Anyother suitable hypertonic media with a osmolarity of at least 300, 310,320, 330, 340, 350, 360, 370, 380, or more mosm may be used in preferredmethods for culturing cells in vitro. These methods may be used toculture cells with or without intracellular sugar. These preferredmedias may also be used before, during, or after storage ofcryopreserved cells.

[0060]FIGS. 9A and 9B are a set of graphs showing the survival of cooledmetaphase II mouse oocytes after overnight culture in modified HTF,hypertonic media as a function of extracellular trehalose concentrationat −15° and −30°, respectively. These experiments were performed using acooling rate of 1° C./min, with or without approximately 0.10 to 0.15 Mintracellular trehalose. Cell viability was assessed using the live/deadassay described herein. Additionally, light microscopy was used tovisually determine whether the viable oocytes had an intact membrane andlacked signs of degeneration and fragmentation. At both −15 and −30° C.,the addition of approximately 0.10 to 0.15 M trehalose inside oocytesdramatically increased the survival rate. Furthermore, at −30 ° C., theabsence of internal trehalose resulted in few viable oocytes afterfreezing. The amount of extracellular trehalose had a dramatic,dose-dependent effect on survival. As the extracellular amount oftrehalose in the freezing solution was increased from 0.15 M(approximately the same amount as the intracellular trehalose) to 0.30 Mor 0.50 M , the survival rate improved from about 18, to 55 or 85%,respectively. This result shows the benefit of using both internal andexternal cryopreservation agents during cooling of cells.

[0061]FIG. 10 shows the survival of cooled metaphase II mouse oocytesafter overnight culture in modified HTF media, isotonic. FIG. 11. showsthe survival of cooled human oocytes after overnight culture in HTFmedia plus 0.1 M extracelluar trehalose. Cell survival was measuredusing the live/dead assay. Light microscopy was also used to visuallydetermine whether the viable oocytes had an intact membrane and lackedsigns of degeneration and fragmentation. The bracket labeled Tg′ in FIG.10 and FIG. 11 shows possible long-term storage temperatures whentrehalose is present both intra- and extracellularly. In FIG. 10,oocytes were cooled at a very slow cooling rate to an intermediatetemperature (−60° C.). Oocytes with no trehalose resulted in a 0%survival rate. Oocytes loaded with 0.50M extracellular trehaloseexperienced some improvement over the control, but suffered a steadydecrease in the survival rate as the temperature decreased. Oocytesloaded with 0.15M intracellular and 0.50M extracellular trehalose, onthe other hand, maintained a survival rate between 80 and 100%. Incomparison with the control and with oocytes in an extracellulartrehalose solution, oocytes containing intracellular trehaloseexperienced a significant increase in survival rate.

[0062]FIG. 12A is a graph with multiple curves that illustrate the watercontent of mouse oocytes as a function of temperature for differentcooling rates, based on the following well-established equations for therate of water transport during freezing (Karlsson et al., HumanReproduction 11:1296-1305, 1996; Toner et al., J. of Membrane Biology115:261-272, 1990).$\frac{V}{T} = {\frac{LpRT}{{Bv}_{w}}{A\left( {{\ln \quad a_{w}^{ex}} - {\ln \quad a_{w}^{in}}} \right)}}$

[0063] where R is the gas constant; T is the temperature; B is thecooling rate; v_(w) is the partial molar volume of water, and a_(w)^(ex) and a_(w) ^(in) in are the water activities in the external andintracellular solutions, respectively. Lp is the water permeabilitygiven by${Lp} = {{Lpg}\quad {\exp \left\lbrack {{- \frac{ELp}{R}}\left( {\frac{1}{T} - \frac{1}{T_{R}}} \right)} \right\rbrack}}$

[0064] where Lpg is the reference water permeability at T_(R); ELp isthe activation energy or temperature dependence of water permeability,and T_(R) is the reference temperature (typically, 0° C.).

[0065] As illustrated in these curves, the extent of dehydration ofoocytes depends on the cooling rate. For example, very slow coolingrates may result in excess dehydration of oocytes. Alternatively, veryfast cooling rates (e.g. 30 or 60° C/min), may result in minimaldehydration. Thus, intermediate cooling rates, such as those between 0.1and 5° C./min, are preferable. For these rates, the water transportmodel predicts that the intracellular water volume will initiallydecrease and then asymptoticly approach a constant value. As illustratedin this figure, water permeability is an exponential function oftemperature with an activation energy, ELp, of 14.5 kcal/mol for mouseoocytes. Thus, as the temperature is lowered during freezing, Lpdecreases precipitously and reaches almost zero for temperatures below−50° C./min. Due to the similarities between the temperature dependenceof Lp (i.e., the value of ELp) for mouse oocytes and oocytes from othermammals, such as rats, bovine, and humans, similar dehydration behavioris expected for other oocytes. For example, ELp is 14.70 kcal/mol forhuman oocytes, compared to 14.5 kcal/mol for mouse oocytes (Paynter etal., Human Reproduction 14: 2338-2342, 1999).

[0066]FIG. 12B is a graph that illustrates the calculated dehydrationtime necessary for the volume of an oocyte to be reduced by 50% at agiven temperature. This graph was generated using the water transportequations listed above and clearly indicates that water transport issignificantly reduced at low temperatures, especially at temperaturesbelow −50° C. Thus, cells are preferably cooled using the methods of theinvention to a final temperature of at least −50, −40, −30, −20, or −10°C. to allow dehydration to continue at a significant rate. Afterdehydration is complete, the cells may be stored at this temperature orat a lower temperature to maintain the cells in a glass state until theyare needed.

[0067]FIG. 13 is a graph with a set of curves showing the percentage ofoocytes that have intracellular ice at various temperatures. For thisassay, standard cryomicroscopy procedures were used to cool oocytes at arate of 3.5° C./min (Cosman et al., Cryo-Letters 10:17-38, 1989). Theoocytes were observed using a cryomicroscope to determine whetherintracellular ice had formed. The presence of intracellular ice waseasily detected based on the black color that appeared due to the lightscattered by the ice crystals. This assay was used to test controloocytes incubated in isotonic media alone, oocytes incubated in mediawith 0.1 M extracellular trehalose, and oocytes injected with 0.1 Mtrehalose and incubated in media with 0.1 M extracellular trehalose. Asillustrated in FIG. 13, extracellular trehalose reduced the incidence ofintracellular ice formation in oocytes. The frequency of intracellularice was further reduced by the presence of intracellular trehalose inaddition to the extracellular trehalose. If desired, the amount ofintracellular ice may be further reduced by increasing the concentrationof intracellular or extracellular sugar that is used. The ability oftrehalose to significantly reduce internal ice allows faster coolingrates. In the field of cryobiology, it has been established that thefaster the cooling rate without formation of lethal intracellular ice,the greater the chance of cell survival.

[0068]FIG. 14 is a table that lists sugars with a glass transitiontemperature of greater than −55° C. As illustrated in this table, sugarswith higher molecular weights tend to have higher glass transitiontemperatures. Linear polymers also tend to have higher glass transitiontemperatures than branched polymers of the same molecular weight.Comparing linear and cyclic α-(1→4)-linked glucose hexamers, a cyclicoligomer (cyclodextrin, Tg′=−9° C.) had a higher glass transitiontemperature than a linear oligomer (maltohexaose, Tg′=−14.5° C.) (Levineand Slade, supra).

[0069] A graph of Tg′ as a function of molecular weight for some sugarsand traditional cryoprotectants is shown in FIG. 15. In a morecomprehensive figure in Levine and Slade (supra) for 84 small sugars,the monotonic relationship between increasing Tg′ and molecular weightyielded a linear correlation of r=−0.93. Thus, for sugars with amolecular weight of at least 120 daltons, their Tg′ is at least −50° C.

Other Embodiments

[0070] From the foregoing description, it will be apparent thatvariations and modifications may be made to the invention describedherein to adopt it to various usages and conditions. Such embodimentsare also within the scope of the following claims.

[0071] All publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

What is claimed is:
 1. A method for treating a living cell, said methodcomprising the steps of: (a) microinjecting into the cytoplasm of saidcell a protective agent which (i) comprises a sugar, (ii) issubstantially non-permeating with respect to mammalian cell membranes,and (iii) maintains the viability of said cell such that it can bestored in a temporarily dormant state and substantially restored to anactive state; (b) treating said cell to cause it to enter said dormantstate; and (c) storing said cell in said dormant state.
 2. The method ofclaim 1, further comprising the step of (d) treating said cell torestore it to an active state.
 3. The method of claim 1, wherein saidcell is a mammalian cell.
 4. The method of claim 3, wherein said cell isan oocyte.
 5. The method of claim 3, wherein said cell is an epithelialcell, neural cell, epidermal cell, keratinocyte, hematopoietic cell,melanocyte, chondrocyte, B-cell, T-cell, erythrocyte, macrophage,monocyte, fibroblast, muscle cell, embryonic stem cell, or adult stemcell.
 6. The method of claim 1, further comprising contacting said cellwith an extracellular protective agent that is substantiallynon-permeating with respect to mammalian cell membranes and thatstabilizes the cell membrane of said cell.
 7. The method of claim 1,wherein said protective agent comprises at least one sugar selected fromthe group consisting of sucrose, trehalose, fructose, dextran, andraffinose.
 8. The method of claim 1, wherein said protective agentcomprises at least one sugar selected from the group consisting ofglucose, sorbitol, mannitol, lactose, maltose, and stachyose.
 9. Themethod of claim 1, wherein said protective agent comprises at least onesugar with a glass transition temperature greater than −50° C.
 10. Themethod of claim 9, wherein said protective agent comprises at least onesugar with a glass transition temperature greater than −30o C.
 11. Themethod of claim 1, wherein said protective agent comprises at least onesugar with a molecular weight greater than 120 daltons.
 12. The methodof claim 1, wherein said protective agent comprises at least one sugarwith a glass transition temperature greater than −30° C. and a molecularweight greater than 120 daltons.
 13. The method of claim 1, wherein saidprotective agent comprises a glycolipid or a glycoprotein that comprisesat least one sugar moiety derived from a sugar with a glass transitiontemperature greater than −50° C.
 14. The method of claim 1, wherein thecytoplasmic concentration of said sugar is less than or equal to about1.0 M following step (a) and prior to step (b).
 15. The method of claim14, wherein the cytoplasmic concentration of said sugar is less than orequal to about 0.2 M following step (a) and prior to step (b).
 16. Themethod of claim 6, wherein said extracellular protective agent comprisesan extracellular sugar.
 17. The method of claim 16, wherein said cell ismaintained in a liquid medium, and wherein the extracellularconcentration of said extracellular sugar is less than or equal to about1.0 M following dilution into said liquid medium.
 18. The method ofclaim 17, wherein the extracellular concentration of said extracellularsugar is less than or equal to about 0.2 M following dilution into saidliquid medium.
 19. The method of claim 16, wherein said cell ismaintained on a solid medium, and wherein the concentration of saidextracellular sugar is less than or equal to about 1.0 M followingadministration to said cell.
 20. The method of claim 19, wherein theconcentration of said extracellular sugar is less than or equal to about0.2 M following administration to said cell.
 21. The method of claim 1,wherein step (b) comprises freezing said cell to a cryogenictemperature.
 22. The method of claim 21, wherein said cell is plungefrozen.
 23. The method of claim 21, wherein step (d) comprises thawingsaid cell.
 24. The method of claim 1, wherein step (b) comprises dryingsaid cell to a level sufficient to permit dry storage.
 25. The method ofclaim 24, wherein step (b) comprises freeze drying said cell.
 26. Themethod of claim 24, wherein step (b) comprises vacuum or convectivedrying said cell.
 27. The method of claim 24, wherein step (d) comprisesrehydrating said cell.
 28. The method of claim 1, wherein only saidprotective agent is employed.
 29. The method of claim 1, wherein priorto step (a), said cell is maintained in a hypertonic medium having anosmolarity greater than 300 mosm.
 30. The method of claim 2, whereinfollowing step (d), said cell is cultured in a hypertonic medium havingan osmolarity greater than 300 mosm.
 31. The method of claim 1, whereina penetrating cryoprotectant mixture is added to said preservationagent.
 32. A method of culturing a cell in vitro, comprising incubatingsaid cell in a hypertonic medium having an osmolarity greater than 300mosm.
 33. The method of claim 32, wherein the osmolarity of said mediumis greater than 320 mosm.